Oncolytic HSV1 vectors and methods of using the same

ABSTRACT

Malignant tumors that are resistant to conventional therapies represent significant therapeutic challenges. An embodiment of the present invention provides an oncolytic virus capable of killing target cells, such as tumor cells. In various embodiments presented herein, the oncolytic viruses described herein are suitable for treatment of several types of cancer, including glioblastoma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Phase Entry of International PatentApplication No. PCT/US2013/070087 filed on Nov. 14, 2013 which claimsbenefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent ApplicationSer. No. 61/726,318 filed on Nov. 14, 2012, the contents of which areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under R21NS0632901,1P01CA163205, and P01CA069246 awarded by National Institutes of Health.The government has certain rights in the invention.

SEQUENCE LISTING

The sequence listing of the present application has been submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “043214-076071-PCT_SL”, creation date of May 13, 2015 and asize of 14,073 bytes. The sequence listing submitted via EFS-Web is partof the specification and is herein incorporated by reference in itsentirety.

FIELD OF INVENTION

The present invention is directed to the fields of virology, cancerbiology, and medicine. More particularly, it concerns compositions andmethods of treating cancer of the brain in a patient using oncolyticherpes simplex virus 1 (HSV-1) armed with therapeutic genes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 14, 2013, isnamed 043214-076071-PCT_SL.txt and is 14,073 bytes in size.

BACKGROUND

Malignant tumors that are intrinsically resistant to conventionaltherapies represent significant therapeutic challenges. Such malignanttumors include, but are not limited to malignant gliomas and recurrentsystemic solid tumors such as lung cancer. Malignant gliomas are themost abundant primary brain tumors, having an annual incidence of 6.4cases per 100,000 (CBTRUS, 2002-2003). These neurologically devastatingtumors are the most common subtype of primary brain tumors and are oneof the deadliest human cancers. In the most aggressive cancermanifestation, glioblastoma multiforme (GBM), median survival durationfor patients is 14 months, despite maximum treatment efforts. Aprototypic disease, malignant glioma is inherently resistant to currenttreatment regimens. In fact, in approximately ⅓ of patients with GBM thetumor will continue to grow despite treatment with radiation andchemotherapy. Median survival even with aggressive treatment includingsurgery, radiation, and chemotherapy is less than 1 year (Schiffer,1998). Because few good treatment options are available for many ofthese refractory tumors, the exploration of novel and innovativetherapeutic approaches is important.

Gene therapy is a promising treatment for tumors, including gliomas, andthe identification of genetic abnormalities contributing to malignanciesis providing important information to aid in the design of genetherapies. Genetic abnormalities indicated in the progression of tumorsinclude the inactivation of tumor suppressor genes and theoverexpression of numerous growth factors and oncogenes. Tumor treatmentmay be accomplished by supplying a polynucleotide encoding a therapeuticpolypeptide or other therapeutic that targets the mutations andresulting aberrant physiologies of tumors. It is these mutations andaberrant physiologies that distinguish tumor cells from normal cells. Atumor-selective virus is an especially promising tool for gene therapy,and recent advances in the knowledge of how viruses replicate have beenused to design tumor-selective oncolytic viruses.

In gliomas, several kinds of conditionally replication competent viruseshave been shown to be useful in animal models, for example: reovirusesthat can replicate selectively in tumors with an activated ras pathway(Coffey et al., 1998); genetically altered herpes simplex viruses(Martuza et al., 1991; Mineta et al., 1995; Andreanski et al., 1997),including those that can be activated by the different expression ofproteins in normal and cancer cells (Chase et al., 1998); and mutantadenoviruses that are unable to express the E1B55 kDa protein and areused to treat p53-mutant tumors (Bischof et al., 1996; Heise et al.,1997; Freytag et al., 1998; Kim et al., 1998). Taken together, thesereports confirm the relevance of oncolytic viruses (OVs) as anti-canceragents. In all three systems, the goal is the intratumoral spread of thevirus and the ability to selectively kill cancer cells. Along withdirectly killing the cancers cells, agents that can also influence themicroenvironment surrounding the tumor may enhance the therapeuticeffect of the OV.

Replication selective oncolytic viruses have shown great promise asanti-tumor agents for solid tumors. The viruses have been constructedgenetically so that they are able to preferentially replicate withintumor cells, while being at least somewhat restricted in their abilityto replicate in normal cells. The principal anti-tumor mechanism ofoncolytic viruses is through a direct cytopathic effect as theypropagate and spread from initially infected tumor cells to surroundingtumor cells, achieving a larger volume of distribution and anticancereffects. Oncolytic herpes simplex viruses (HSVs) were initially designedand constructed for the treatment of brain tumors. Subsequently, theyhave been found to be effective in a variety of other human solidtumors, including breast, prostate, lung, ovarian, colon and livercancers. The safety of oncolytic HSVs has also been extensively testedin mice and primates, which are extremely sensitive to HSV.

HSV-1 based oncolytic viruses are particularly promising because of: (1)their ability to infect a wide variety of tumors; (2) their inherentcytolytic nature; (3) their well-characterized large genome (152 Kb)that provides ample opportunity for genetic manipulations wherein manyof the non-essential genes can be replaced by therapeutic genes; (4)their ability to remain as episomes that avoid insertional mutagenesisin infected cells; and (5) the availability of anti-herpetic drugs tokeep in check possible undesirable replication.

Despite encouraging preclinical studies, results from early clinicaltrials have suggested that most of the current versions of oncolyticviruses, although acceptably safe, may only have limited anti-tumoractivity on their own. While not wishing to be bound by any oneparticular theory, one of the main reasons for the sub-optimal oncolyticefficacy is probably because viral gene deletions that confer tumorselectivity also result in reduced potency of the virus in tumors. Forexample, the complete elimination of endogenous γ34.5 function from HSV,one of the common approaches for the construction of oncolytic HSV,significantly reduces viral replication potential and therefore maycompromise the ability of the virus to spread within the targeted tumors(Kramm et al., 1997).

Considering the limited effective treatment options available forcertain types of cancer, including certain types of brain cancer, thereremains a need in the art for improved oncolytic viruses.

SUMMARY OF THE INVENTION

In various embodiments, the invention teaches an oncolytic expressionvector including a nucleic acid that includes a nucleotide sequenceencoding a GADD34 protein, or a biologically active portion thereof,wherein said nucleotide sequence is operably linked to an expressioncontrol sequence. In certain embodiments, the vector is a modifiedherpes simplex virus. In some embodiments, the modified herpes simplexvirus is a herpes simplex virus deficient for a γ₁34.5 gene. In certainembodiments, the nucleotide sequence includes SEQ ID NO: 1 or adegenerate variant thereof. In various embodiments, the nucleotidesequence includes SEQ ID NO: 2 or a degenerate variant thereof. In someembodiments, the expression control sequence includes a nestin promoteror a biologically active portion thereof. In various embodiments, theexpression control sequence includes SEQ ID NO: 3 or a degeneratevariant thereof.

In various embodiments, the invention teaches a method of killingintracranial tumor cells in a subject. In some embodiments, the methodincludes introducing into the vicinity of the tumor cells an oncolyticexpression vector, said oncolytic expression vector including a nucleicacid that includes a nucleotide sequence encoding GADD34, or abiologically active portion thereof, wherein said nucleotide sequence isoperably linked to an expression control sequence. In certainembodiments, the oncolytic expression vector is a modified herpes virus.In some embodiments, the modified herpes virus is deficient for a γ₁34.5gene. In some embodiments, the nucleotide sequence includes SEQ ID NO: 1or a degenerate variant thereof. In some embodiments, the nucleotidesequence includes SEQ ID NO: 2 or of a degenerate variant thereof. Incertain embodiments, the expression control sequence includes a nestinpromoter or a biologically active portion thereof. In certainembodiments, the expression control sequence includes SEQ ID NO: 3. Insome embodiments, the method also includes the step of mixing apharmacologically acceptable carrier with the oncolytic expressionvector prior to the introducing step. In certain embodiments, the tumorcells include a glioblastoma cell. In some embodiments, the tumor cellsinclude a cancer stem cell. In various embodiments, the subject is amammal. In some embodiments, the subject is a human.

In various embodiments, the invention teaches an oncolytic expressionvector for use in the treatment of intracranial tumor cells in asubject, said oncolytic expression vector including a nucleic acid thatincludes a nucleotide sequence encoding GADD34, or a biologically activeportion thereof, wherein said nucleotide sequence is operably linked toan expression control sequence. In some embodiments, the oncolyticexpression vector is a modified herpes virus. In some embodiments, themodified herpes virus is deficient for a γ₁34.5 gene. In certainembodiments, the nucleotide sequence includes SEQ ID NO: 1 or adegenerate variant thereof. In various embodiments, the nucleotidesequence includes SEQ ID NO: 2 or a degenerate variant thereof. In someembodiments, the expression control sequence includes a nestin promoteror a biologically active portion thereof. In some embodiments, theexpression control sequence includes SEQ ID NO: 3. In certainembodiments, the tumor cells include a glioblastoma cell. In certainembodiments, the tumor cells include a cancer stem cell. In variousembodiments, the subject is a mammal. In some embodiments, the subjectis a human.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in the referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1A demonstrates, in accordance with an embodiment of the invention,a schematic representation of ICP34.5. The multifaceted HSV-1 ICP34.5protein, encoded by the γ₁34.5 gene, is typically considered to be 248a.a., although the lengths are varied by strains. The c-terminal GADD34homology domain contains PP1a (a.a. 193 to 195) and eIF2α bindingdomains (a.a. 233-248) to mediate dephosphorylation of phosphorylatedeIF2α, the phosphorylated state of which suppresses translation via thePKR-mediated innate immune response pathway in response to HSV1infection, resulting in significantly reducing viral productivity. TheBeclin1 binding domain (a.a. 68-87) exerts an inhibitory effect againstautophagy via interaction with Beclin-1, which is an essential proteinfor the autophagy process. This ICP34.5-mediated antagonism of beclin1autophagy function is important for viral neurovirulence.

FIGS. 1B and 1C demonstrate, in accordance with an embodiment of theinvention, an HSV-1 recombinant virus containing a mutation in ICP34.5that abrogates binding to Beclin 1 is neuroattenuated in vivo. (B)Survival of C57BL/6J mice infected intracerebrally with 5×10⁵ pfu ofeither HSV-1 34.5Δ68-87 or its marker rescue (HSV-1 34.5Δ68-87R).Results shown represent survival data combined from four independentinfections. Similar results were observed in each experiment. (C) Viralreplication of HSV-1 34.5Δ68-87 and HSV-1 34.5Δ68-87R in brain tissue ofinfected mice at indicated time after infection. Lower limit ofdetection=1.7. Data shown represent mean±SEM geometric titer for sevento ten mice per experimental group per time point.

FIGS. 1D and 1E demonstrate, in accordance with an embodiment of theinvention, a comparison of the ICP34.5 effect in glioma therapy usingoHSV1s. (D) U251, U87dEGFR, U138, MGH238, T98G, and Gli36d5 glioma cellswere infected with 10⁴ pfu of either rHsvQ1 (γ₁34.5 gene deletion) orrQNestin34.5 (γ₁34.5 gene under the control of a nestin transcriptionalpromoter). Titers of each sample were determined 3 days after infection.Titers of rQNestin34.5 were higher than those of rHsvQ1 in all gliomacell lines (*, P<0.05, Student's t test) (E), human astrocytes wereinfected with either rQNestin34.5 or rHsvQ1 and titers were determined 3days later. There was no statistically significant difference in values(P>0.1).

FIG. 2 demonstrates, in accordance with an embodiment of the invention,a schematic representation of GADD34. The human GADD34 gene is mapped onchromosome 19q13.2, a region containing a cluster of DNA repair genes,and has 3 exons and spans at least 2.6 kb (see Hollander et al.Mammalian GADD34, an apoptosis- and DNA damage-inducible gene. J BiolChem. 1997 May 23; 272(21): 13731-7). GADD34ΔNter lacks the first 174amino acid (a.a.) or 522 bp of DNA sequence, where the ER-localizationdomain and Ubiquitin (Ub) proteasome degradation targeting region exist.GADD34 protein has several domains, MEMB at the N-terminal region, PESTrepeat in the middle, and KVRF (SEQ ID NO: 7) and RARA (SEQ ID NO: 8)sequences containing ICP34.5 homologous domain at the C-terminal region.MEMB domain contributes to ER-membrane association and the lack of thisregion or mutations in this helical domain impair localization to the ERand also mitochondria. The regions rich in proline, glutamic acid,serine and threonine (PEST) are generally known to serve as proteolyticsignals, but deletion of internal PEST repeats had no impact on GADD34stability, however it modulated the binding and activity of PP1 todephosphorylate eIF2α. A bipartite carboxyl terminal domain encompassesthe highly conserved KVRF sequence (SEQ ID NO: 7) (a.a. 555-558), acanonical PP1-binding motif, and RARA sequence (SEQ ID NO: 8), which isalso required for PP1 binding. GADD34 and SNF5/INI1, which is also knownas a tumor suppressor protein and a component of the hSWI/SNF chromatinremodeling complex, can coexist in a trimeric complex with chimericleukemic HRX fusion proteins, leading to inhibition of GADD34-mediatedapoptosis in acute leukemia. And this KVRF (SEQ ID NO: 7) containingregion is the site that interacts with SNF5 protein, which alsoindependently binds to the PP1 catalytic subunit, forming a stabletrimeric complex of SNF5-PP1-GADD34. Therefore, GADD34 mediates growthsuppression and functions as a tumor suppressor, at least in part,through its interaction with SNF5, which may also function as aregulatory subunit of PPI. The N-terminal peptides (1-60 a.a.) exhibitdegradation signal peptides with a half-life of <2 h via 26S proteasome.

FIG. 3 demonstrates, in accordance with an embodiment of the invention,a schematic map of pTnestin-GADD34ΔNter. Full-length or N-terminaltruncated GADD34 gene was inserted into NcoI/HpaI sites ofpTnestin-luc-b vector that inserts the nestin-hsp68 promoter-enhancerelement into pTransfer, by ligating the fragment obtained by enzymaticdigestion of blunt-ended BstXI/XhoI or HpaI/XhoI of a pOTB7-GADD34 (SEQID NO: 1), respectively. Resulting constructs were calledpTnestin-GADD34 or pTnestin-GADD34ΔNter. Those shuttle vectors were usedto make fHsvQuik-based oHSV1 as described in FIG. 4.

FIG. 4A demonstrates, in accordance with an embodiment of the invention,a schematic strategy of cloning MGH1 genome into a BAC vector. MGH1 is astrain F-derived HSV-1 mutant that possesses deletions in both copies ofthe g 1 34.5 gene and a lacZ insertion at the UL 39 locus. A UL39-targeting BAC plasmid, pRBAC-D6GpA-FL, was constructed so that itcontains two homology arms that can recombine with the viral genomeupstream and downstream of the lacZ insertion within the UL 39 locus.The construct also has (1) a set of two recombination sequences, loxPand FRT sites, flanking the BAC backbone, (2) an EGFP expressioncassette which is inserted in-frame downstream of the truncated UL 39coding sequence and (3) an RFP expression cassette within the BACbackbone. The linearized pRBAC-D6GpA-FL DNA and intact MGH1 virion DNAwere co-transfected into Vero cells, and recombinant virus that carriesthe BAC sequence (MGH1-BAC) was isolated. The transgene cassette ofinterest (X) is first cloned into a pTransfer shuttle plasmid and theresulting plasmid (pTransfer-X) is electroporated together with anFLP-expressing helper plasmid into bacteria carrying fHsvQuik-1 BACplasmid. Co-integrants of the pTransfer-X and fHsvQuik-1 fused at theFRT sites (fHsvQ1-X) can be readily obtained by selection with Cm andAmp at 43° C. The fHsvQ1-X has the transgene cassette inserted at the UL39 locus and two unidirectional loxP sites are now flanking all theprocaryotic plasmid backbones as well as the RFP marker gene. Uponco-transfection of the fHsvQ1-X and a Cre-expressing helper plasmid intoVero cells, the procaryotic plasmid backbones, together with the RFPexpression cassette, can be excised through Cre-mediated site-specificrecombination. As a result, recombinant HSV vectors with the transgenecassette (rHsvQ1-X) can be rescued.

FIG. 4B demonstrates, in accordance with an embodiment of the invention,novel oncolytic HSV1, NG34 and NG34C. Using a BAC-based oncolytic HSV1vector (ΔICP6 and ΔICP34.5 product genes) system that is called as“HSVQuik system” (see Gene Ther 13(8):705-14, 2006, which isincorporated herein by reference in its entirety), two oHSV1 vectorswere developed, which insert full-length and N-terminal truncated humangene derived GADD34 gene under control of nestin promoter that is activein glioma cells, where previously developed rQNesting34.5 harbors onecopy of gamma(1)34.5 gene (see Cancer Res. 2005 April 1; 65(7):2832-9,which is incorporated herein by reference in its entirety).

FIG. 5A demonstrates, in accordance with an embodiment of the invention,phosphorylation of eIF2α was suppressed in response to NG34 and NG34Cinfection in glioma. Western Blotting of cell lysates at 16 hpost-infection of oHSV1 at MOI of 0.1 showed that while ICP34.5-nullmutant rHSVQ1 (ΔICP6, ΔICP34.5) infection resulted in strongphosphorylation of eIF2α to suppress translational initiation, NG34 andNG34C viruses, as well as rQNestin34.5 reversed the phosphorylationlevels. Antibodies against HSV-1, eIF2α, phospho(Ser51)-eIF2α, PP, ICP4and αTubulin were used in the assay.

FIG. 5B demonstrates, in accordance with an embodiment of the invention,GADD34 expression upon NG34 and NG34C infection. Full-length GADD34 orN-terminal region truncated GADD34 was overexpressed from cells inresponse to NG34 and NG34C infection, respectively, at 16 hpost-infection at MOI of 0.1, while wild-type HSV1 strain F and otherGADD34 noncoding oHSV1 infection did not cause GADD34 expression.Western blots were performed using antibodies against GADD34, ICP4 andαTubulin.

FIG. 6A demonstrates, in accordance with an embodiment of the invention,the rapid viral life cycle of NG34C in glioma cells. Four differentoHSV1 were infected into U251 cells for 24, 48 and 72 hours beforecollecting cells and media. Titrations were performed on Vero cells tomeasure plaque forming units (PFU).

FIG. 6B demonstrates, in accordance with an embodiment of the invention,phase contrast microscopic images taken at 48 h post-infection torepresent the plaque sizes of each oHSV1 in U251 cells.

FIG. 7 demonstrates, in accordance with an embodiment of the invention,equivalent infectivity of NG34C as rQNestin34.5 in primary glioblastomacells isolated form patients. rQNestin34.5 and NG34C were infected into5 different GBMs cultured in serum-free Neurobasal media on PLL/laminincoated plates for three days, subsequent to the titration in Vero. N=3

FIG. 8 demonstrates, in accordance with an embodiment of the invention,NG34C replicated more in lower oxygen condition cultured primaryglioblastoma cells. rQNestin34.5 and NG34C at MOI of 0.1 were infectedin 4 different GBMs cultured in serum-free Neurobasal media onPLL/laminin coated plates under normoxia (21% O2) or lower physiologicaloxygen condition (5% O2) for three days, subsequent to the titration inVero. N=3

FIG. 9 demonstrates, in accordance with an embodiment of the invention,F strain-permissive primary normal tissue cells are resistant to NG34Cinfection. Glioma cell lines (U87dEGFR and U251), and primary humantissues (Astrocyte, Hepatocyte, Smooth Muscle and Lung fibroblast) wereinfected with wild-type HSV1 (F strain) and mutant HSV-1 (rHSVQ1,rQNestin34.5 and NG34C). These wild-type HSV1-permissive primary tissuesdidn't support the replication of mutant HSV1.

FIG. 10 demonstrates, in accordance with an embodiment of the invention,NG34C virus infected cells produced more progeny virus in medium (sup)than intracellular or on cellular membranes. Progeny virus yields weresimilar among rQNestin34.5, NG34 or NG34C, and 2-digits lower by rHSVQ1virus as expected. However, NG34C progeny were higher in medium thanin/on infected cells, while other oHSV1s were lower in medium,suggesting that NG34C is capable of more rapid distribution among cellsthan NG34 or rQNestin34.5 while total viral production doesn't changemuch between those oHSV1.

FIG. 11 demonstrates, in accordance with an embodiment of the invention,NG34C is more resistant to 1% hypoxia microenvironment, compared torQNestin34.5 and NG34. Under severely lower oxygen conditions or hypoxiaconditions (1% O2, 5% CO2 and 94% N2), oHSV1 replication was limited andthe effect of ICP34.5 or GADD34 was not seen in rQNestin34.5 or NG34infected U251 cells, when viral yields were compared to ICP34.5-nullrHSVQ1 virus. In contrast, NG34C yields were still lower in hypoxia thanthat in normoxia (21%), but higher than other viruses.

FIG. 12A demonstrates, in accordance with an embodiment of theinvention, cytotoxicity of oHSV1 infection using U87ΔEGFR glioma cells.Serial dilutions of various HSV1 (F, hrR3, rHSVQ1, NG34C) from 0 to 1PFU/cell were used to infected U87ΔdEGFR on a 96-well plate for 5 daysbefore measuring quantitatively lactate dehydrogenase (LDH), a stablecytosolic enzyme that is released upon cell lysis, using CytoTox 96cytotoxicity assay kit (Promega). N=4

FIG. 12B data from panel (a) was plotted in the upper graph andmedian-effect dose (Dm) values (as MOI) were calculated in the table. m:a measurement of the sigmoidicity of the dose-effect curve, r: thelinear correction coefficient of the median-effect plot.

FIGS. 13A and 13B demonstrate, in accordance with an embodiment of theinvention, evaluation of the synergistic cytotoxic effect of oHSV1 andTMZ To investigate the synergism of oHSV1 with standard chemotherapeuticdrug, temozolomide (TMZ), oHSV1 were diluted serially in 96-well plateand mixed with/without EC30 dose of TMZ (66 μM for U87ΔEGFR) beforeplating cells at numbers of 5,000. After 5 days, released LDH wasmeasured using CytoTox 96 cytotoxicity assay kit. Synergism wascalculated using formula of Chou-Talalay's combination indices(CalcuSyn, BioSoft Inc.). CI: combination index.

FIG. 13C demonstrates, in accordance with an embodiment of theinvention, data from the tables of 13A and 13B were plotted. Less than 1indicates synergism and more than 1 indicates antagonism. NG34 andrQNestin34.5 show more synergism at Dm values and NG34C showed more thanDm values. NG34, NG34C and rQNestin34.5 represented a broader range ofsynergistic effects with TMZ than rHSVQ or wild-type F or hrR3.

FIG. 13D demonstrates, in accordance with an embodiment of theinvention, a graphic representation of the range of synergistic effects.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3^(rd) ed., J. Wiley & Sons (New York, N.Y. 2001); March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^(th)ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russell,Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, certain terms are defined below.

In some embodiments, the numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the application are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the application are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable.

“Mammal,” as used herein, refers to a member of the class Mammalia,including, without limitation, humans, as well as nonhuman primates suchas chimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, newborn subjects and infant subjects, as well as fetuses, whethermale or female, are intended to be included within the scope of thisterm.

The term “vector,” as used herein, refers to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). One of skill inthe art would be well equipped to construct a vector through standardrecombinant techniques (see, for example, Maniatis et al., 1988 andAusubel et al., 1994, both of which are incorporated herein byreference). Additionally, the techniques described herein anddemonstrated in the referenced figures are also instructive with regardto effective vector construction.

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

The term “promoter,” as used herein, refers to a nucleic acid sequencethat regulates, either directly or indirectly, the transcription of acorresponding nucleic acid coding sequence to which it is operablylinked. The promoter may function alone to regulate transcription, or,in some cases, may act in concert with one or more other regulatorysequences such as an enhancer or silencer to regulate transcription ofthe gene of interest. The promoter comprises a DNA regulatory sequence,wherein the regulatory sequence is derived from a gene, which is capableof binding RNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. A promoter generally comprises asequence that functions to position the start site for RNA synthesis.The best-known example of this is the TATA box, but in some promoterslacking a TATA box, such as, for example, the promoter for the mammalianterminal deoxynucleotidyl transferase gene and the promoter for the SV40late genes, a discrete element overlying the start site itself helps tofix the place of initiation. Additional promoter elements regulate thefrequency of transcriptional initiation. Typically, these are located inthe region 30-110 bp upstream of the start site, although a number ofpromoters have been shown to contain functional elements downstream ofthe start site as well. To bring a coding sequence “under the controlof” a promoter, one can position the 5′ end of the transcriptioninitiation site of the transcriptional reading frame “downstream” of(i.e., 3′ of) the chosen promoter. The “upstream” promoter stimulatestranscription of the DNA and promotes expression of the encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. Depending on the promoter used, individualelements can function either cooperatively or independently to activatetranscription. The promoters described herein may or may not be used inconjunction with an “enhancer,” which refers to a cis-acting regulatorysequence involved in the transcriptional activation of a nucleic acidsequence, such as those for the genes, or portions or functionalequivalents thereof, listed herein.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages may begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include thebeta-lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. As demonstrated herein, in some embodiments, a nestin promoteris used to drive expression of the gene of interest. In addition toproducing nucleic acid sequences of promoters and enhancerssynthetically, sequences may be produced using recombinant cloningand/or nucleic acid amplification technology, in connection with thecompositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and5,928,906, each incorporated herein by reference). Furthermore, it iscontemplated the control sequences that direct transcription and/orexpression of sequences within non-nuclear organelles such asmitochondria, chloroplasts, and the like, can be employed as well.

The term “recombinant HSV-1 vector,” as used herein, defines arecombinant HSV-1 vector comprising: (a) the DNA of, or correspondingto, at least a portion of the genome of an HSV-1 that is capable oftransducing into a target cell at least one selected gene and is capableof promoting replication and packaging; and (b) at least one selectedgene (or transgene) operatively linked to at least one regulatorysequence directing its expression, the gene flanked by the DNA of (a)and capable of expression in the target cell in vivo or in vitro. Thus,a “recombinant HSV” (rHSV) means HSV that has been genetically altered,e.g., by the addition or insertion of a selected gene.

A “gene,” or a “sequence which encodes” a particular protein, is anucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of one or more appropriate regulatorysequences. A gene of interest can include, but is no way limited to,cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA,and even synthetic DNA sequences. A transcription termination sequencewill usually be located 3′ to the gene sequence. Typically, apolyadenylation signal is provided to terminate transcription of genesinserted into a recombinant virus.

The term “polypeptide” or “protein,” as used herein, means a polymer ofamino acids joined in a specific sequence by peptide bonds. As usedherein, the term “amino acid” refers to either the D or L stereoisomerform of the amino acid, unless otherwise specifically designated.

The term “transgene” refers to a particular nucleic acid sequenceencoding a polypeptide or a portion of a polypeptide to be expressed ina cell into which the nucleic acid sequence is inserted. The term“transgene” is meant to include (1) a nucleic acid sequence that is notnaturally found in the cell (i.e., a heterologous nucleic acidsequence); (2) a nucleic acid sequence that is a mutant form of anucleic acid sequence naturally found in the cell into which it has beeninserted; (3) a nucleic acid sequence that serves to add additionalcopies of the same (i.e., homologous) or a similar nucleic acid sequencenaturally occurring in the cell into which it has been inserted; or (4)a silent naturally occurring or homologous nucleic acid sequence whoseexpression is induced in the cell into which it has been inserted. A“mutant form” or “modified nucleic acid” or “modified nucleotide”sequence means a sequence that contains one or more nucleotides that aredifferent from the wild-type or naturally occurring sequence, i.e., themutant nucleic acid sequence contains one or more nucleotidesubstitutions, deletions, and/or insertions. In some cases, the gene ofinterest may also include a sequence encoding a leader peptide or signalsequence such that the transgene product may be secreted from the cell.

As used herein, the term “transfection” refers to the uptake of foreignDNA by a mammalian cell. A cell has been “transfected” when exogenousDNA has been introduced inside the cell membrane. A number oftransfection techniques are known in the art. See, Graham et al. (1973)Virology, 52:456; and Sambrook et al. (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Laboratories, New York. Suchtechniques can be used to introduce one or more exogenous DNA moieties,such as a viral vector and other nucleic acid molecules, into suitablehost cells. The term refers to both stable and transient uptake of thegenetic material.

The term “oncolytic activity,” as used herein, refers to cytotoxiceffects in vitro and/or in vivo exerted on tumor cells without anyappreciable or significant deleterious effects to normal cells under thesame conditions. The cytotoxic effects under in vitro conditions aredetected by various means as known in prior art, for example, bystaining with a selective stain for dead cells, by inhibition of DNAsynthesis, or by apoptosis. Detection of the cytotoxic effects under invivo conditions is performed by methods known in the art.

A “biologically active” portion of a molecule, as used herein, refers toa portion of a larger molecule that can perform a similar function asthe larger molecule. Merely by way of non-limiting example, abiologically active portion of a promoter is any portion of a promoterthat retains the ability to influence gene expression, even if onlyslightly. Similarly, a biologically active portion of a protein is anyportion of a protein which retains the ability to perform one or morebiological functions of the full-length protein (e.g. binding withanother molecule, phosphorylation, etc.), even if only slightly.

With the aforementioned preliminary descriptions and definitions inmind, additional background is provided herein below to provide contextfor the genesis and development of the inventive vectors, compositionsand methods described herein.

Current mutant HSV-1 vectors that target malignant glioma are based onthe two deletion mutant genes, ICP6 (U_(L)39 gene product), the largesubunit of HSV-1 ribonucleotide reductase (RR), and ICP34.5 (34.5 geneproduct), a multifunctional protein that is also related toneurovirulence. While the lack of ICP6 restricts virus replication tonon-dividing cells but allows replication to continue in cells withdefects in the p16 tumor suppressor pathway, deletions of both γ₂ 34.5genes suppresses HSV-1 encephalitis. While not wishing to be bound byany one particular theory, this may be due to ICP34.5's facilitation ofBeclin-1 autophagy function, essential for neurovirulence. Besides thisautophagic inhibitory effect, ICP34.5 also counteracts a host defensemechanism triggered by viral infection. This mechanism activates PKR(double-stranded RNA protein kinase) that then phosphorylates thetranslation factor, eIF2α, leading to translation inhibition. ICP34.5directly binds and activates PP1 (protein phosphatase 1) thatdephosphorylates eIF2α, allowing for viral mRNA translation to continue.Oncolytic HSV-1 with mutated γ34.5 genes (e.g. G207, 1716) has proven tobe safe for administration in humans with gliomas in multiple clinicaltrials, but efficacy has been elusive, probably due to their limitedviral replication.

To overcome this limitation, an HSV1 was previously engineered, whereinthe ICP34.5 gene is under the transcriptional control of the glioma stemcell promoter for nestin. rQNestin34.5 has exhibited increased efficacyin glioma models and currently a phase I clinical trial in adults withglioblastoma is being pursued. However, there remains a potentialconcern with expression of ICP34.5 in the brain, particularly inindividuals (children and young adults) whose brains may still berelatively rich with nestin-positive neural progenitor cells and whereexpression of ICP34.5 could trigger neurovirulence. Expression ofICP34.5 has also been reported to contribute to Alzheimer disease andother neurodegenerative diseases by suppressing autophagy function inneurons. Finally, ICP34.5's autophagy inhibition may reduce thetherapeutic efficacy of conventional chemo- and radiotherapies that relyon autophagy-mediated tumor cell death in apoptosis-resistant malignantgliomas.

Growth_Arrest and DNA-Damage inducible gene 34 (GADD34), also known asPPP1R15A, was discovered by screening a radiation-treated myeloblasticleukemia cell cDNA library. Treatment of various human cell lines withDNA-damaging agents enhanced expression of GADD34. A stress-inducibleGADD34 protein also interacts with PP1 and reverses phosphorylation ofeIF2α, preventing complete shutoff of protein synthesis during stressconditions in the same way as ICP34.5 does, since its c-terminus sharessignificant homology with the C-terminus of GADD34. Apart from enhancedprotein synthesis via the PP1 complex during conditions of cellularstress, GADD34 forms a stable complex with tuberous sclerosis complex(TSC) 1/2, causes TSC dephosphorylation, and inhibits the mTOR signalingpathway. Therefore, GADD34 could be a potential mTOR inhibitor forcancer therapy. In fact, conditionally ectopic GADD34 overexpression inU251 human glioma cells likely induced cell growth delay and senescence(data not shown). This mTOR suppression via TSC1-GADD34 complex can alsoinduce cytoprotective autophagy under the condition of misfolded mutantprotein expression and during starvation. In addition, recently GADD34was reported as a neuroprotective factor in neurodegenerative disease,including Alzheimer's, Parkinson's and prion diseases. Neurodegenerativediseases are associated with the accumulation of misfoldeddisease-specific proteins, triggering the unfolded protein response(UPR) pathway, resulting in the transient shutdown of proteintranslation, through phosphorylation of eIF2 alpha. In prion-diseasedmice, overexpression of GADD34 restored vital translation rates duringprion disease, rescuing synaptic deficits and neuronal loss, therebysignificantly increasing survival.

Because it is a stress inducible factor, turnover of GADD34 protein israpid and the N-terminal peptides (1-60 a.a.) exhibit a degradationsignal peptide (degron) with a half-life of <2 h via 26S proteasome. Ofnote, it was recently reported that phosphorylation of tyrosine-262 alsocontributes to the rate of GADD34 turnover and a non-phosphorylationmutant form (Y262F) displayed a significant increase t_(1/2)>2 hr. Also,the N terminal 180 residues of GADD34 directs the localization to theendoplasmic reticulum (ER) and it targets the alpha isoform of PP1 tothe ER. Interestingly, while this N-terminal truncated form (180-674aa)still retains the capacity to dephosphorylate eIF2 alpha via PP1, itlacks ER specific localization. A truncated mutant (513-674aa), whichencompassed the ICP34.5 homology domain, was exclusively in nucleoli andlacks the capability of dephosphorylation of eIF2 alpha.

Overall, these findings indicate that the use of GADD34 in an oncolyticHSV1 vector can enhance therapeutic efficacy, while reducingcytotoxicity in non-cancer cells. Because of the dual roles of GADD34 ofenhanced protein synthesis via dephosphorylation of eIF2α, and inductionof autophagy via TSC1/mTOR pathway, it could support viral replicationin cancer cells and circumvent the limitation of ICP34.5 expressionleading to possible neurovirulence in response to oHSV1 infection.

An N terminal truncated form (GADD34ΔN) was evaluated, in which thefirst 174 amino acids residues were deleted to prevent rapid degradationof GADD34 protein and to suppress potential ER-stress induction. Toprove the hypothesis that GADD34 expression in the context of oHSVallows for robust replication and cytotoxicity in glioma cells, but notin normal cells, novel HSV1 oncolytic viruses, NG34 and NG34C, weredesigned to express the full length (1-674aa) and the N-terminaltruncated form (175-674aa) of the GADD34 gene under the control of anestin promoter, respectively. A summary of the relevant characteristicsdiscovered, and described above, is provided in Table I.

TABLE I Summary HSV1 wt GADD34 GADD34ΔNter ICP34.5 (1-674aa) (175-674aa)PP1 interaction + + + eIF2α + + + dephosphorylation ER localization − +− Beclin-1 interaction + − − TSC − + + dephosphorylation Proteasomal − +− degradation Autophagy activity Down Up Up neurovilurence + No reportNo report Neuroprotective effect Down Up Up

With the foregoing findings in mind, certain embodiments of theinvention teach an oncolytic expression vector. In some embodiments, theoncolytic expression vector includes a nucleic acid including anucleotide sequence of interest which encodes the GADD34 protein, abiologically active portion thereof (such as a truncated versionthereof), or a functional equivalent thereof. In certain embodiments,the nucleotide sequence of interest is operably linked to an expressioncontrol sequence. In some embodiments, the expression control sequenceincluded in the oncolytic expression vector is a promoter. In someembodiments, the promoter is a nestin promoter. In certain embodiments,the oncolytic vector is a recombinant HSV-1 vector. In some embodiments,the recombinant HSV-1 vector is deficient for the γ₁34.5 gene. Invarious embodiments, the nucleotide sequence encoding the GADD34 proteinincludes SEQ ID NO: 1 or a degenerate variant of SEQ ID NO: 1. In someembodiments, the nucleotide sequence encodes a truncated GADD34 gene. Insome embodiments, the truncated GADD34 gene is GADD34C. GADD34C isencoded by SEQ ID NO: 2. One of skill in the art would readilyappreciate that a degenerate variant of SEQ ID NO: 2 could be used as analternative to SEQ ID NO: 2. In some embodiments, the expression controlsequence includes the nestin promoter, as demonstrated in SEQ ID NO: 3.One of skill in the art would readily appreciate that a modified versionof SEQ ID NO: 3 could also be used, so long as it retains similarbiological activity. Merely by way of non-limiting example, the nestin2^(nd) intron sequence (enhancer) represented in SEQ ID NO: 4, and thehsp68 minimum promoter represented in SEQ ID NO: 5, could be used aloneor combined when designing various constructs contemplated herein. Insome embodiments, the nestin enhancer element may be operably linked toa heat shock protein 68 (hsp68) minimum promoter to drive the expressionof GADD34 delta-Nter, as demonstrated in SEQ ID NO: 6. In someembodiments, alternative or additional expression control sequences maybe incorporated into the oncolytic expression vectors to initiate orinfluence the expression of any of the aforementioned nucleotidesequences of interest. Merely by way of non-limiting examples, anytumor- or tissue-specific promoter or other expression controlsequences, such as microRNA target sequences, may be used. Examples ofspecific promoters included, but are not limited to, CEA for coloncancer cells, Muc1 for breast cancer cells, Myb1 for all cancer cells,Tyrosinase for melanoma cells, PSA for prostate cancer cells. Examplesof miR translational control sequences include, but are not limited to:miR128 or miR124 to differentiate glioma cells from normal neural cells.In some embodiments, alternative oncolytic expression vectors, asidefrom HSV-1, can be used to facilitate the expression of GADD34, GADD34C,or one or more biologically active portions thereof.

In various embodiments, the present invention provides a method fortreating a neoplastic disease in a subject. In certain embodiments, themethod includes administering to the subject a therapeutically effectiveamount of an expression vector with oncolytic activity. In someembodiments, the expression vector is a tumor-specific conditionalreplication vector. In some embodiments, the vector is a recombinantHSV-1 vector. In some embodiments, the recombinant HSV-1 vector includesone or more copies of a DNA sequence of interest encoding a GADD34protein, one or more portions of the GADD34 protein, or a functionalequivalent of the GADD34 protein. In some embodiments, the DNA sequenceof interest is GADD34C. In some embodiments, one or more of theaforementioned DNA sequences is operably linked to an expression controlsequence, which may include any of those expression control sequencesdescribed herein above. In some embodiments, the expression controlsequence is a promoter configured to facilitate expression of the DNAsequence. In some embodiments the promoter is a nestin promoter. In someembodiments, an alternative promoter, such as any of those describedherein, or designed according to the methods described herein, can beused. In some embodiments, the neoplastic disease that is treated iscancer. In some embodiments, the cancer is brain cancer. Merely by wayof non-limiting examples, the types of brain cancer that can be treatedmay include glioblastoma, anaplastic astrocytoma, astrocytoma, pilocyticastrocytoma, diffuse intrinsic pontine glioma, oligodendroglioma,anaplastic oligodendroglioma, mixed oligo-astrocytoma, and pendymoma. Insome embodiments, cancer stem cells are treated with the inventivemethod. In some embodiments, the subject treated is a mammal. In certainembodiments, the subject treated is a human.

Methods of treating any of the neoplastic diseases described herein,including brain cancer, may include administration of the compounds ofexemplary embodiments as a single active agent, or in combination withadditional methods of treatment including, but not limited to, stemcell-based therapy, immunotherapy, radiation therapy, therapy withimmunosuppressive agents, chemotherapeutic or anti-proliferative agents,including cytokines. The methods of treatment of the invention may be inparallel to, prior to, or following additional methods of treatment.

As indicated above, the GADD34 or GADD34C DNA sequences, or portionsthereof that can be used in conjunction with the inventive constructsand methods described herein include those that have been modified. Whenexpressed, modified DNA sequences include those that can result in aminoacid substitutions (e.g., at one or more of the important amino acidresidues) of the GADD34 or GADD34C protein. A modified GADD34 or GADD34Cprotein can have altered biological activity (increased or decreased) orsubstantially the same activity (functionally equivalent), as comparedto unmodified GADD34 or GADD34C protein, especially with regard tofacilitating dephosphorylation of eIF2 alpha.

The promoter operably linked to the gene of interest, which can includebut is not limited to the GADD34 gene, N-terminal truncated GADD34 gene,otherwise modified GADD34 gene, or functional equivalent of the GADD34gene, used in the vectors, compositions and methods described herein, ispreferably a promoter that can drive expression of the gene of interestin a cancer cell. In preferred embodiments, the promoter used in theinventive vectors, compositions and methods facilitates levels ofexpression of the gene of interest that are sufficient to result in (1)reduced phosphorylation of eIF2 alpha, and/or (2) significant viralreplication, and/or (3) significant oncolysis, such that sometherapeutic benefit results. “Therapeutic benefit,” as used herein,includes any decrease in cancer cell number, cancer cell proliferationrate, or metastasis. In some embodiments, the promoter used hereinfacilitates selective or increased expression of the associated gene ofinterest in one or more cancer cell type of interest, compared to anormal cell.

The term “operably linked,” as used herein, refers to the arrangement ofvarious nucleic acid molecule elements relative to each other such thatthe elements are functionally connected and are able to interact witheach other. Such elements may include, without limitation, a promoter,an enhancer, a polyadenylation sequence, one or more introns and/orexons, and a coding sequence of a gene of interest to be expressed. Thenucleic acid sequence elements, when operably linked, can act togetherto modulate the activity of one another, and ultimately may affect thelevel of expression of the gene of interest, including any of thoseencoded by the sequences described above.

The nucleic acid sequence of the “full length” GADD34 gene used in theexperiments reported herein and described in the referenced figures isprovided herein as SEQ ID NO: 1. The nucleic acid sequence of theN-terminal truncated GADD34 gene is provided herein as SEQ ID NO: 2. Thenucleic acid sequence of the nestin promoter control sequence used inthe experiments reported herein is provided as SEQ ID NO: 3. Althoughthese specific sequences are provided, the nucleic acid molecules usedin the inventive vectors, compositions and methods are not limitedstrictly to molecules including the sequences set forth as SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.Rather, specific embodiments encompass nucleic acid molecules carryingmodifications such as substitutions, small deletions, insertions, orinversions. Included in the invention are nucleic acid molecules, thenucleotide sequence of which is at least 95% identical (e.g., at least96%, 97%, 98%, or 99% identical) to the nucleotide sequence shown as SEQID NOS: 1, 2, 3, 4, 5, and 6 in the Sequence Listing.

Also included in the invention is a nucleic acid molecule that has anucleotide sequence which is a degenerate variant of a nucleic aciddisclosed herein, e.g., SEQ ID NOS: 1, 2, 3, 4, 5, and 6. A sequentialgrouping of three nucleotides, a “codon,” encodes one amino acid. Sincethere are 64 possible codons, but only 20 natural amino acids, mostamino acids are encoded by more than one codon. This natural“degeneracy” or “redundancy” of the genetic code is well known in theart. It will thus be appreciated that the nucleic acid sequences shownin the Sequence Listing provide only an example within a large butdefinite group of nucleic acid sequences that will encode thepolypeptides as described above.

Importantly, the vectors of the embodiments described herein may beuseful for the introduction of additional genes in gene therapy. Thus,for example, the HSV vectors of this invention may contain one or moreadditional exogenous gene for the expression of a protein effective inregulating the cell cycle, such as p53, Rb, or mitosin, or abiologically active variant thereof, or in inducing cell death, such asthe conditional suicide gene thymidine kinase, the latter must be usedin conjunction with a thymidine kinase metabolite in order to beeffective, or any other anti-tumor gene, such as for example a toxin.

When used pharmaceutically, oncolytic vector embodiments discussedherein can be combined with various pharmaceutically acceptablecarriers. Suitable pharmaceutically acceptable carriers are well knownto those of skill in the art. The compositions can then be administeredtherapeutically or prophylactically, in effective amounts, described ingreater detail below.

As used herein, the term “therapeutically effective amount” is intendedto mean the amount of vector which exerts oncolytic activity, causingattenuation or inhibition of tumor cell proliferation, leading to tumorregression. An effective amount will vary, depending upon the pathologyor condition to be treated, by the patient and his or her status, andother factors well known to those of skill in the art. Effective amountsare easily determined by those of skill in the art. In some embodimentsa therapeutic range is from 10³ to 10¹² plaque forming units introducedonce. In some embodiments a therapeutic dose in the aforementionedtherapeutic range is administered at an interval from every day to everymonth via the intratumoral, intrathecal, convection-enhanced,intravenous or intra-arterial route.

Although certain routes of administration are provided in the foregoingdescription, according to the invention, any suitable route ofadministration of the vectors may be adapted, and therefore the routesof administration described above are not intended to be limiting.Routes of administration may including but are not limited to,intravenous, oral, buccal, intranasal, inhalation, topical applicationto a mucosal membrane or injection, including intratumoral, intradermal,intrathecal, intracisternal, intralesional or any other type ofinjection. Administration can be effected continuously or intermittentlyand will vary with the subject and the condition to be treated. One ofskill in the art would readily appreciate that the various routes ofadministration described herein would allow for the inventive vectors orcompositions to be delivered on, in, or near the tumor or targetedcancer cells. One of skill in the art would also readily appreciate thatvarious routes of administration described herein will allow for thevectors and compositions described herein to be delivered to a region inthe vicinity of the tumor or individual cells to be treated. “In thevicinity” can include any tissue or bodily fluid in the subject that isin sufficiently close proximity to the tumor or individual cancer cellssuch that at least a portion of the vectors or compositions administeredto the subject reach their intended targets and exert their therapeuticeffects.

Pharmaceutically acceptable carriers are well known in the art andinclude aqueous solutions such as physiologically buffered saline orother solvents or vehicles such as glycols, glycerol, vegetable oils(e.g., olive oil) or injectable organic esters. A pharmaceuticallyacceptable carrier can be used to administer the compositions of theinvention to a cell in vitro or to a subject in vivo. A pharmaceuticallyacceptable carrier can contain a physiologically acceptable compoundthat acts, for example, to stabilize the composition or to increase theabsorption of the agent. A physiologically acceptable compound caninclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. Other physiologically acceptable compounds include wettingagents, emulsifying agents, dispersing agents or preservatives, whichare particularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art would knowthat the choice of a pharmaceutically acceptable carrier, including aphysiologically acceptable compound, depends, for example, on the routeof administration of the polypeptide. For example, a physiologicallyacceptable compound such as aluminum monosterate or gelatin isparticularly useful as a delaying agent, which prolongs the rate ofabsorption of a pharmaceutical composition administered to a subject.Further examples of carriers, stabilizers or adjutants can be found inMartin, Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton,1975), incorporated herein by reference.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those skilled in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Vector Constructs of the NG34 and NG34C Oncolytic Viruses

The HSVQuik method was employed to engineer HSV1 vectors as describedherein and previously (see Terada et al. Development of a rapid methodto generate multiple oncolytic HSV vectors and their in vivo evaluationusing syngeneic mouse tumor models. Gene Ther 2006 April; 13(8):705-14,which is incorporated herein by reference in its entirety). First, afull-length or N-terminal truncated GADD34 gene was inserted into theNcoI/HpaI sites of pTnestin-luc-b vector that inserts the nestin-hsp68promoter-enhancer element into pTransfer, by ligating the fragmentobtained by enzymatic digestion of blunt-ended BstXI/XhoI or HpaI/XhoIof a pOTB7-GADD34 (SEQ ID NO: 1) respectively (FIG. 3). These shuttlevectors were used to transform E. coli carrying the bacterial artificialchromosome (BAC) called fHsvQuik2, which has two flp recombination FRTsites within this UL39 locus, but lacks the EGFP gene from flsvQuik1(see Terada et al. Development of a rapid method to generate multipleoncolytic HSV vectors and their in vivo evaluation using syngeneic mousetumor models. Gene Ther. 2006 April; 13(8):705-14, which is incorporatedherein by reference in its entirety). FLP-FRT mediated site-specificrecombination between the shuttle vectors and fHsvQuik2 BAC resulted infHsvQ2-nestin-GADD34 and flsvQ2-nestin-GADD34ΔN BAC vectors,respectively (FIG. 4). Vero cells were transfected with these BACs and apc-nCre, a Cre recombinase-expression vector to remove all theprokaryotic sequences from the shuttle vector flanking loxP sites. Theresulting HSV1 viruses NG34 (containing full length GADD34) and NG34C(truncated GADD34) were generated and packaged in these Vero cells.

Example 2 Description of HSVQuik Vector System

The HsvQuik technology was developed as a novel BAC (bacterialartificial chromosome)-based method for the generation of oncolyticHSV-1. It takes advantage of relatively rapid and easy methods ofconventional cloning by combining two sequential, site-specificrecombination systems (Flp-FRT and Cre-loxP). The basic backbone forfHsvQuik vectors has been the genome of an oncolytic HSV-1 designated asMGH1, consisting of a double-mutant oncolytic HSV-1 (F strain) that hasa lacZ gene insertional mutant UL39 (encoding a large subunit ofribonucleotide reductase, ICP6) and deletions of diploid γ₁34.5 genesencoding the neurovirulence factor ICP34.5 responsible for encephalitis.To stably maintain this large HSV-1 genome in E. coli, the lacZ:UL39locus of the MGH1 genome was also engineered to express additional genesencoding for the fluorescent markers, DsRed1 (for fHsvQuik-1 and -2) andEGFP (for fHsvQuik-1). The fHsvQuik BAC genome is not directlymanipulated, rather its engineering with additional desired sequences isaccomplished via a shuttle vector (pTransfer) that has been engineeredwith multiple cloning sites (MCSs) to insert gene(s) of interest withdesired regulatory sequences (e.g. transcriptional regulatory elements).pTransfer contains an FRT site to allow direct site-specific integrationinto the fHsvQuik BAC DNA in Escherichia coli (E. coli) withoutenzymatic manipulation (e.g. DNA ligation). Because the R6Kγ replicationorigin of the pTransfer plasmid depends on E. coli strains that possessthe pir gene, R6Kγ ori doesn't interfere with the bacterial replicationorigin of the fHsvQuik BAC in DH10B (pir⁻) E. coli strains after Flp-FRTrecombination. To remove the BAC sequences from the fHsvQuik DNA, theCre-loxP recombination technique is used. The resultant recombinantHSV-1 clones forming individual plaques are distinguished fromnon-recombinant BAC-containing HSV-1 clones by RFP fluorescence sincethe CMV promoter-driving DsRed1 gene is also excised along with the BACsequences during Cre-LoxP recombination.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (for example, “such as”) provided withrespect to certain embodiments herein is intended merely to betterilluminate the application and does not pose a limitation on the scopeof the application otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element essential tothe practice of the application.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

In closing, it is to be understood that the embodiments of theapplication disclosed herein are illustrative of the principles of theembodiments of the application. Other modifications that can be employedcan be within the scope of the application. Thus, by way of example, butnot of limitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

What is claimed is:
 1. An oncolytic herpes simplex virus deficient for aγ₁34.5 gene comprising a nucleic acid comprising: a) a nucleotidesequence encoding N-terminal truncated human GADD34 protein, wherein thenucleotide sequence is set forth in SEQ ID NO: 2, wherein saidnucleotide sequence is operably linked to an expression controlsequence.
 2. The oncolytic herpes simplex virus of claim 1, wherein theexpression control sequence comprises a nestin promoter or abiologically active portion thereof.
 3. The oncolytic herpes simplexvirus of claim 1, wherein the expression control sequence comprises SEQID NO:
 3. 4. The oncolytic herpes simplex virus of claim 1, wherein theexpression control sequence comprises a tumor specific promoter.
 5. Theoncolytic herpes simplex virus of claim 4, wherein the tumor specificpromoter is specific for colon cancer cells, breast cancer cells,melanoma cells, or protstate cancer cells.
 6. The oncolytic herpessimplex virus of claim 4, wherein the tumor specific promoter isselected from the group consisting of a CEA promoter, a Muc1 promoter, aMyb1 promoter, a Tyrosinase promoter, and a PSA promoter.